Cytogenetic Methods

Cytogenetic methods

František Šťáhlavský

1 Cytogenetic is a branch of genetics that is concerned with the study of the structure and function of the cell, especially the chromosomes.

1842 – first observations of chromosomes 1888 – used term chromosome (chroma=colour , soma=body ) 1902-04 – chromosomal inheritance theory

1950s – progress in methods (hypotonization,… 1956 – final determination of 2n in human 1968 – banding techniques 1990s – FISH techniques

During this lecture, we will focus on Cytogenetic techniques. The cytogenetic is relatively old branch of science analyzing mainly the chromosomes and this research is closely related to the development of microscopes during the nineteenth century. The first microscoped chromosomes were observed like small dark dots inside the cells. Shortly after these findings the chromosomal inheritance theory was presented which means that the chromosomes hold genetic information and transfer this information equally to the new cells during the cell division. However, an early analysis used only histological sections that don’t provide a good resolution for chromosome observation. Therefore, it was not easy to count the diploid numbers and very often it was incorrect. This was improved during the fifties when new cytogenetic methods were developed. Since then it was possible to specify the number and morphology of chromosomes more precisely. For example, the correct number of chromosomes in human cell was specified in 1956. The second half of the last century became a cytogenetics boom. The new banding techniques were developed that enabled to identify homology of chromosomes, even between the species. The new era represents the implementation of Fluorescent in situ hybridization which is better known as FISH technigue. This technique enables to identify the location of specific genes. But even the standard cytogenetic techniques may also help you to identify changes of genome reorganization. Moreover, these techniques may be very fast and cheap. So, they are still very useful.

2 Functions of chromosomes (2x histons H2A, H2B, H3 a H4) - spatial distribution of genes

- transport of genetic information during cell division

- crossing-over during meiosis, EUCHROMATIN x HETEROCHROMATIN new genetic combinations genetic activity no genetic activity

Drosophila melanogaster (2n = 8)

On this slide, we need to repeat basic information about the chromosomes. Chromosomes are not only the DNA but they also contain different proteins (mainly several histones) that enable the spatial organization of the DNA and regulate also the activity of the genes. The chromosomes are usually condensed and visible as interphase nuclei in the cells. The interphase nucleus may seem to be a chaotic structure, but it is obviously not. The FISH techniques show that every chromosome has a specific position inside this interphase nucleus. Moreover, the chromosomes have not the same value regarding their genetic content. We distinguish Euchromatin with genetic activity and Heterochromatin with no genetic activity. Here are three main functions of chromosomes. The first is the specific localization of the genes. During the rearrangements of chromosomes, the genes may change their position and may be transferred to the new areas that may change their transcription activity. The second, chromosomes also guarantee equal separation of genetic information to new cells during cell division. And third during the meiosis, the crossing over between homologous chromosomes forms new genetic variability.

3 Melters et al. 2012

1 2 3X

H holocentrics scorpion Tityus bahiensis (2n=5-19) Pseudoscorpion: Olpium turcicum: 2n = 7, X0

We distinguish two different types of chromosomes that differ in the organization of kinetochores. This structure is located on the surface of chromosomes and makes the connection to microtubules of spindle apparatus that guarantee equal separation of chromosomes to the new sister cells during cell division. In the monocentric chromosomes, the kinetochore is located only in a small area that we name centromere. This area is often visible as a constriction on the chromosome, so-called primary constriction. The second type is the holocentric chromosomes. In this case, the kinetochore is spread on the surface of the chromosome. This organization is beneficial when some fractures happen because than both fragments of chromosome include kinetochore for connection to microtubules and still, they may be equally separated during cell division. While in the monocentric chromosomes fragments without kinetochores are eliminated. It is still disputable which type of chromosomes is ancestral because both types are distributed equally all-over different organisms.

4 Monocentric chromosomes

M SM ST T

Holocentric (holokinetic) chromosomes

www.metasystems-international.com/ikaros

www.lucia.cz

In monocentric chromosomes, we recognize four morphological types according to the position of the centromere. It is easy to qualify them as the ratio of long and short arms. The holocentric chromosomes have not any visible constriction. So, we don’t have a chance to specify the morphology of these chromosomes, we can measure only the length. It is a reason why it is more complicated to identify homologs in holocentrics without additional banding methods. You can use automatic software to sort and categorize the chromosomes, for example, IKAROS and LUCIA. However, these systems are developed mainly for human diagnostics and so they are not cheap.

5 http://imagej.nih.gov/ij/ http://rsb.info.nih.gov/ij/plugins/levan/levan.html

http://www.drawid.xyz

If you want to describe the karyotype you also can use free software. The main opportunity is plugin LEVAN for ImajeJ that sort the chromosomes according to your measurements of the arms of chromosomes. Similarly, you can use also DRAWID that enables also to create the ideogram.

6 Karyogram of scirpion Bothriurus rochensis

Karyogram of Astyanax fasciatus deduced after conventional Idiogram (ideogram) Giemsa staining and double FISH using 5S (green) and 18S of scorpion Tityus trivittatus rDNA (red) probes.

Here is not any mandatory style of visualization of karyotypes. You can find mainly two different types in the articles. The first type sorts the chromosome pairs according to the size and the morphology is specified usually separately in the text or table. The second type of visualization sorts the chromosome pairs firstly according to morphology than according to the size. You can find also ideograms (or idiograms) that represent schematic visualization of chromosomes. They may contain specific characteristics like the location of different bands of genes.

7 number of chromosomes

from 2n=2 to 2n = 446 Plebicula atlantica Parascaris univalens, ants Myrmecia pilosula, M. croslandi, (males n=1)

Márquez-Corro et al. 2018

The knowledge about the karyotypes is very useful, especially for species delimitation. It is known, that karyotypes of organisms differ sometimes very distinctly, even between closely related species. The variability of the basic characteristic such as the number of chromosomes is very wide within animals. The diploid numbers range from two chromosomes in some ants and mites to more than four hundred in one butterfly. It should be noted that ants have haplodiploidy, it means that males are haploid. So, the lowest number of chromosomes is in fact only one chromosome. The higher limit of chromosomes may be even higher in some polyploids. As I already told you, we recognize monocentric and holocentric chromosomes. The fragmentation is more probable in holocentric chromosomes. It is also the case of lycaenid butterfly Plebicula atlantica (it was demonstrated that this species is not polyploid but the chromosomes are fragmented). For many years it was presented that the holocentrics are more variable owing to the frequent fragmentation. Interestingly, the recent analysis shows that the diversification rates of karyotypes are similar between sister groups with monocentrics and holocentrics.

8 number of chromosomes Modal number of chromosomes Odonata n=13

Lepidoptera n=28-32 spiders

birds n=39-42 Salticidae Diptera n=2-10

different families

Iguanidae

pseudoscorpions

Despite the high variability of karyotypes, some groups may have a very stable number of chromosomes. And this most frequent number is called MODAL NUMBER. The number may be stable in some groups or may change during the evolution and then become constant again (as you can see in family Iguanidae).

9 Zima 2000 Fish app. 30000 species - 1700 karyotyped

http://coleoguy.github.io/karyotypes/

Arachnids Karyotypes http://www.arthropodacytogenetics.bio.br/index.html

For the utilization of the information about the karyotypes for example in the taxonomy, it is very important to know the variability of closely related taxa. It is relatively easy for example in mammals. At this moment we have information about the karyotypes in the majority of mammal species, including also the information about the intraspecific variability (especially in Europe). Unfortunately, we have not so much information in the remaining groups especially in invertebrates. Currently we have several online databases with the karyotypes of amphibians, beetles, polyneoptera, and some arachnids.

10 Euscorpius (Alpiscorpius) germanus group Scorpiones

Now, I would like to show you some results of our team that use the cytogenetic characteristic in species delimitation of scorpions. The members of this arachnid order are very morphological uniform and this group contains many cryptic species. It is for example the case of Euscorpius gemanus, the endemic species group in the Alps. Till the end of the last century, it was recognized as only one species with small morphological variability.

11 16S rRNA Euscorpius (Alpiscorpius) germanus group Scorpiones Gantenbein et al. 2000

Scherabon et al. 2000

One of the first molecular analyses in scorpion using 16S rRNA mitochondrial genes disclosed that Euscorpius germanus represents, in fact, three different cryptic species without clear morphological differences but with the distinct allopatric distribution. These results were based on the genetic distances and we hoped that karyotypes will approve these differences.

12 Euscorpius (Alpiscorpius) germanus 2n=60 group Scorpiones

2n=90

2n=54

2n=46 Cryptocercus

2n=60* 2n=58 2n=92*

2n=88*

Štundlová et al. 2019

Che et al. 2006

However, we found twice more species according to the karyotypes in the Alps. It is clear, that the simple comparison of genetic distances is not the correct approach in this case, because the speciation processes and the history of the species may differ between the areas of the Alps. The scorpions are typical sedentary animals that are not able to move for long distances and the high karyotype diversity is typical for such organisms. Similar results that we obtained in the Alps are known for example also in cockroaches of the genus Cryptocercus without the wings and in several other groups.

13 Euscorpius (Alpiscorpius) germanus 2n=60 group Scorpiones

2n=90

2n=54 chromosome speciation ?

“hybrid-sterility model” predicted that the recombination among rearranged chromosomes in 2n=46 heterokaryotypic hybrids generate unbalanced gametes and thus reduce fertility.

2n=60* 2n=58 2n=92*

2n=88* “suppressed-recombination model” suggest that the rearrangements reduce recombination between chromosomes and lead to the divergence and speciation. Štundlová et al. 2019

Big differences between closely related species led to the hypothesis of the direct impact of chromosomal changes to the speciation. Here are two generally accepted models of chromosome speciation. The hybrid-sterility model predicted that the recombination among rearranged chromosomes in hybrids generate unbalanced gametes and thus reduce fertility. The suppressed-recombination model suggests that the rearrangements reduce recombination between chromosomes and lead to divergence and speciation. However, it is still disputable if the chromosomal changes are reason or consequence of the speciation.

14 types of chromosomal rearrangements

Here are presented the main chromosomal rearrangements that may change the karyotypes. The deletion has a strictly negative effect. On the other hand, duplication may have a positive effect. Some of the rearrangements are not easy to detect, especially paracentric inversions because they do not include centromeres and do not change morphology and the size of chromosomes.

15 types of chromosomal rearrangements

Mitotic metaphase Meiotic pachytene

Scorpion Gint gaitako, 2n=30, Kovařík et al. 2019

Some rearrangements are easy to identify especially during meiosis. For example, here are the holocentric chromosomes of one scorpion species. As I already told you, we don’t recognize the morphology of chromosomes in holocentrics, and in this species, the chromosomes gradually decrease in length. So, it is not easy to find homologs. However, it is easy to find them during pachytene. During this stage, it is clear that this karyotype includes reciprocal translocation.

16 Number fundamental Phillips & Ráb 2001 Centric fusions - Robertsonian translocations or fissions

Mus musculus domesticus

Tandem fusion pseudoscorpions Chthonius (E.) tetrachelatus Chthonius (E.) sp. 1 0,25 2n = 35 2n = 29

0,20 0,20

0,15 0,15

0,10 0,10

0,05 0,05

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 X 1234567891011121314 X 0,00 0,00

The centric fusions or fissions are called Robertsonian translocations. This type of rearrangement changes the number of chromosomes very fast. However, it does not change the number of arms. That is a reason why some analysis specifies also NUMBER FUNDAMENTAL that represents the number of arms. As you can see in the salmon fishes the diploid number differs considerably among the species. However, the number fundamental is not variable. It is evident that the Robertsonian translocations are the main mechanisms of karyotype differentiation in this group. Another specific rearrangement is tandem fusion. This rearrangement changes the length of some chromosome that starts to be longer than the remaining pairs.

17 Mitosis

S - phase DNA synthesis G 2 9 h 4 h 0,9 h

10 h

G 1

If you want to analyze the karyotypes and compare specific markers using cytogenetic techniques, you usually need chromosomes that are clearly visible and separated from each other. Therefore, we usually use the mitotic metaphases. During this phase, the chromosomes are condensed and clearly visible. However, this stage is very short during the cell life. Fortunately, FISH techniques enable us to detect and visualize the specific probes even in the interphase nucleus.

18 Meiosis

Sex chromosome

Especially in invertebrates, we also can analyze the chromosomes during meiosis. In contrast to mitosis, we can observe homology during the first prophase very easily. This enables to detect heteromorphic sex chromosomes much more precisely in comparison to mitotic metaphase.

19 Cytogenetic techniques

The most important – good quality of chromosome preparation

- Dividing cells bone marrow, blood, amniotic fluid, cord blood, tumor, and tissues (including skin, umbilical cord, chorionic villi, liver, and many other organs) In invertebrates very often salivary gland, embryo, testis phytohaemagglutinin

A mitotic inhibitor (colchicine, colcemid) is added to the culture. This stops cell division at mitosis which allows an increased yield of mitotic cells for analysis.

- Hypotonic solution

Potassium chloride (KCl), Citric acid (Na3C6H5O7) - Fixation methanol (or ethanol ) : glacial acetic acid (3:1) Carnoy's fixative - ethanol : chloroform : glacial acetic acid (6:3:1)

allways fresh !! - acid acid alcohol ester - Spreading (good quality of microscope slides !!) „dropping“ „squashing“ „plate spreading“

If you want to use cytogenetic techniques, you always need top quality chromosome preparation. The main premise is to have some tissue with dividing cells. Cell culture is very common in vertebrates. The salivary glands, embryos, or testis are usually used in invertebrates. The chromosome preparation contains three important steps. The first one is the application of the hypotonic solution. During this step, the cells intake the water and the chromosomes become more distant and visible. The cells must be in a hypotonic solution for a very specific time. If they are in the hypotonic solution too short the chromosomes are still close to each other and they are not visible very well. On the other hand, after a long time, the cells may crack and so your chromosomes are lost. The second step is fixation. Always prepare fresh fixative. Otherwise, acetic acid together with alcohol forms ester that may damage the chromosome structure. In the last step you prepare a thin layer of the cells on the microscope slide. You can drop, squash, or evaporate the suspension on this surface. Always use the microscope slides of high quality.

20 Conventional staining – homogeneous staining

Giemsa Number, morfology and size of chromosomes Haematoxylin Acid-Schiff staining Carbol fuchsin

Scorpion: Bothriurus rochensis pseudoscorpions:

0,20 Chthonius (E.) fuscimanus 0,20 Chthonius (E.) tetrachelatus 2n = 35 2n = 35 0,15 0,15

0,10 0,10

0,05 0,05

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 X 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 X 0,00 0,00

0,25 0,25 Chthonius (E.) sp. 2 Chthonius (E.) sp. 1 0,20 0,20 2n = 29 2n = 21 0,15 0,15

0,10 0,10

0,05 0,05

1 2 3 456 7 8 9 10 X 0,00 12 34567891011121314 X 0,00

Šťáhlavský & Král 2004

When you obtain microscope slides with chromosomes you need to visualize them. The fastest and cheapest is conventional staining (e.g. Giemsa). After this staining, you can count the number of chromosomes and specify the morphology and the size. However, it is not sometimes easy to identify the position of the centromere and especially the homology of chromosomes.

21 Conventional staining – homogeneous staining

Giemsa sex chromosomes, rearrangements

mistake

Scorpion: Hottentotta judaicus Harvestmen: Gagrellopsis nodulifera (Gorlov & Tsurusaki 2000) Spiders: Spermophora senoculata Pholcus phalangioides Diguetia albolineata Holocnemus caudatus no heteromorphic bivalent during pachytene

Qumsiyeh et al. 2013

Kral et al. 2006

The problem with homology may produce mistakes. For example, distinct sex chromosomes XY were described in scorpion Hottentota judaicus. However, the sex chromosomes are not known in any scorpion species at this moment. It is evident that the authors erroneously build the karyogram in this species. They overlooked that there is no such heteromorphic bivalent during pachytene of meiosis. On the other hand, the careful analysis of the meiosis may disclose specific chromosomal rearrangements and complicated sex chromosome systems only with conventional staining.

22 Conventional staining – homogeneous staining

Giemsa

B chromosomes

Hervestmen Metagagrella tenuipes Tsurusaki 1993

Acanthocephalus lucii. Chromosome sets of 5 female individuals. (a) 2n = 6 + XX; (b–e) 2n = 6 + XX + 2–5B (Špakulová et al. 2002)

Another complication represents B chromosomes. These types of chromosomes (also called supernumerary chromosomes) do not contain important genetic information for the organisms, and they are distributed randomly to new cells. That is a reason the number of B chromosomes may differ between the cells of a single individual and may reach the number of the rest of chromosomes. In such cases, they have usually a negative effect on the individual.

23 Conventional staining – homogeneous staining

Ancestral state Mesquite ChromEvol v. 2.0 http://www.mesquiteproject.org/ http://www.tau.ac.il/~itaym ay/cp/chrom Evol/

fissions

fusions

Mycetophylax ants fusions

fusions

fissions

Micolino et al. 2020 Sci. Rep.

If you have information about the karyotypes in more species, you can try to identify the ancestral state and the main trends in the karyotype evolution for the specific lineages. You may use for example free software ChromEvol or Mesquite to assess the ancestral state of chromosome number.

24 Selective staining – for specifique regions, large blocks

C- banding - constitutive heterochromatin

0.2 M HCl for 20-45 min (depurination) Rinse with DI water 4% Ba(OH)2 (barium hydroxid) at 60 °C (denaturation) Rinse with DI water 2x SSC at 60 °C for 20-75min (renaturation) Rinse with DI water Podysma krylonensis Bugrov et al. 2004

Ovis orientalis anatolica Arslan & Zima 2004

The staining methods are used for the identification of the homology among the chromosomes. One of the most frequently used is C-banding that may be either used in vertebrates or invertebrates. This technique identifies constitutive heterochromatin that is usually localized in centromeres. Moreover, sex chromosome Y and B chromosomes very often contain large blocks of heterochromatin and some specific interstitial blocks may be observed in some autosomes.

25 Selective staining – for specifique regions, large blocks

Ag-NOR staining - NOR = Nucleolar Organizing Region

The region contains several tandem copies of ribosomal DNA genes.

- 1 g of AgNO3 in 1 mL of 0.02 g sodium citrate (C6H5Na3O7·2 H2O) per 500 mL distilled water, adjusted to pH 3.0 with formic acid. - Add 1-2 drops of the above solution onto the slides and place a cover slip over the preparation. - Incubate slides in a moist chamber at 55–60°C for app. 30 min.

Ovis orientalis anatolica Arslan & Zima 2004

Very popular is silver staining. This method visualizes proteins connected to the Nucleolar Organizing Regions. However, this technique visualizes only the Nucleolar Organizing Regions (NORs) that were metabolically active during the previous cell division. So, the number of NORs may be underestimated with this technique. That is a reason why the FISH techniques with a specific probe for rRNA genes are more frequent now.

26 Selective staining – for specifique regions, large blocks

G- banding - obtained by the action of trypsin (10-20s at room temperature in a fresh 0.25% trypsin and than washed in PBS to block the action of trypsin) similar pattern also in Q-banding (satained by quinacrin)

Sorex araneus 2n=20-33 FN=40 Chrom. races=72

Sorex araneus

The G banding is possible to induce only in mammals. It is a very specific banding of chromosomes that enables identification of homology very precisely. Owing to this banding techniques they were described more than 70 chromosomal races in common shrew that differ mainly by Robertsonian translocations (the fundamental number is the same for all races).

27 Selective staining – for specifique regions, large blocks

Comparison of G-banded chromosomes of Sorex minutus and S. granarius Biltueva et al. 2011

The G bands may also identify homology between different species.

28 2n=48

2n=46

And G banding also enables to identify chromosomal rearrangements in karyotype evolution very precisely. Here is, for example, the reconstruction of chromosomal changes among apes.

29 Selective staining – for specifique regions, large blocks

R- banding – bands reverse to G-banding - the thermal denaturation of chromosomes (30-90 minutes at 87°C)

G- bands R- bands AT rich regions GC rich regions

Fluorochrome staining AT rich regions: DAPI (4',6-diamidin-2-fenylindol), chinakrin, Hoechst 33258 GC rich regions: chromomycin A3, mithramycin, olivomycin

Here are also some other stainings that produce specific bands on chromosomes. They usually visualize AT rich or GC rich regions. They are not so frequently used as the previous ones.

30 Transmission electron microscopy (TEM)

Ultrastructure of pairing of the X univalents with acrocentric chromosomes of the trivalent, Malthonica ferruginea male. (Král 2007)

To analyze the chromosome structures, we don’t use only the light or fluorescent microscopes. We can also use transmission electron microscopy. This technique is not used for the description of karyotypes but enables mainly to identify how the chromosomes pair during meiosis. For example, in spider Malthonica ferruginea transmission electron microscopy helped to identify cryptic sex chromosomes that only pair during pachytene with previously known sex chromosomes.

31 FISH - Fluorescence In Situ Hybridization

indirect labeling direct labeling

The main technique that is now frequently used in cytogenetics is Fluorescent in Situ Hybridization. This method enables to identify specific DNA sequences directly on chromosomes. The preparation of the chromosomes is the same as for the other methods. You only need to prepare a specific probe that you can hybridize to the target DNA. Usually, you can prepare the probe using PCR. During PCR you can use nucleotides connected to fluorochromes. This type represents direct labeling. Indirect labeling represents the case when the nucleotides are connected to the proteins and the signal is later visualized with antigens connected with fluorochromes. In this case, the signal is usually stronger. Fluorochromes are chemicals that can absorb energy from an excitation source and emit photons at a longer wavelength. If you use a different filter you can use several probes simultaneously.

32 NICK translation - DNA Polymerase I is used to replace some of the nucleotides of a DNA sequence with their labeled analogues

Primed in situ Labelling (PRINS)

Here are the methods for labeling the probes. The first method I already described in the previous slide. It means that you can add labeled nucleotides directly into the PCR. The second method replaces some nucleotides of obtained DNA sequences later using NICK translation kit. Another chance is also making PCR with labeled nucleotides directly on the surface of chromosome slides. It is Primed in situ labeling.

33 telomere Insects (TTAGG)n Types of probes Vertebrates, Anellida, Mollusca (TTAGGG)n Nematoda (TTAGGC)n - Satelite DNA - centromeric - telomeric

Ravatsos et al. 2015 - painting probes

- locus specific

Pokorná et al. 2011

In general, we can distinguish three types of probes. Very popular are probes for centromeric or telomeric regions. These types of probes may clearly identify fusions of chromosomes during karyotype evolution. The painting probes visualize the whole chromosomes. And the locus-specific probes detect the position of specific genes on chromosomes.

34 Centromere-FISH (ACM-FISH) Immuno-FISH armFISH Locked Nucleic Acids (LNAs)-FISH Catalyzed Reporter Deposition-FISH (CARD-FISH) Multiplex (M)-FISH Cellular Compartment Analysis of Temporal (Cat) Multilocus or ML-FISH Activity by Fish (catFISH) Premature Chromosome Condensation (PCC)-FISH Cytochalasin B (CB-FISH) Peptide Nucleic Acid (PNA)-FISH Chromosome Orientation (CO)-FISH Quantitative-FISH (Q-FISH) Combined Binary Ratio (COBRA)-FISH Quantum Dot (QD)-FISH Chromosome Orientation and Direction (COD)- Rainbow-FISH FISH Raman-FISH Combinatorial Oligonucleotide (COMBO)-FISH Replicative Detargeting FISH (ReD-FISH) Comet-FISH Reverse-FISH Cryo-FISH Recognition of Individual Genes (RING)-FISH Double Fusion FISH (D-FISH) RNA-FISH DNA Breakage Detection FISH (DBD-FISH) Cross Species Color Banding (Rx)-FISH e-FISH Split-Signal FISH Fiber-FISH Stellaris RNA FISH (Single-Molecule RNA FISH) Flow-FISH T-FISH Fusion-Signal FISH 3-D FISH Halo-FISH Zoo-FISH Harlequin-FISH

Currently we have many modifications of FISH. Many of them are used mainly for human diagnostics.

35 locus specific

Ribosomal DNA (rDNA) - loci encoding 5S and 45S (18S-5.8S-28S) rRNAs 18S rDNA probe

Borba et al. 2014 Nguyen et al. 2010

One of the most frequently used FISH detects rRNA genes. FISH is more specific than the silver staining. The identification of rRNA genes is a little bit easier in comparison to other genes because rRNA genes are organized in repeated clusters. The knowledge of rRNA genes enables to identify chromosomal rearrangements in monocentric as well as holocentric chromosomes.

36 locus specific

Nemacheilidae

Sember et al. 2015

Ribosomal DNA (rDNA) - loci encoding 5S and 45S (18S-5.8S-28S) rRNAs

It is interesting that 5S and 45S (18S-5.8S-28S) RNA genes have different evolution and frequently is localized on different chromosomes.

37 locus specific http://www.animalrdnadatabase.com

Sochorová et al. 2018

Ribosomal DNA (rDNA) - loci encoding 5S and 45S (18S-5.8S-28S) rRNAs

Number of 5S and 45S rDNA sites in different animal taxa

54 loci 45S/2C

De Barros et al. 2017

It is available an online database and review about rRNA genes in animals. The maximum numbers of 45S sites were 54 loci, found in fish Schizodon fasciatus and the maximum numbers of 5S sites were found 74 sites in the neotropical lizards from the Teiidae family, species Kentropyx pelviceps. However, about 60% karyotypes had only a single 45S and 5S locus.

38 locus specific http://www.animalrdnadatabase.com

Sochorová et al. 2018

Ribosomal DNA (rDNA) - loci encoding 5S and 45S (18S-5.8S-28S) rRNAs

Position of rDNA sites on chromosomes

54 loci 45S/2C

De Barros et al. 2017

Concerning the position of rRNA genes, it is usually terminal in 45S in the majority of groups. Only in arthropods, it is the location more equally also in interstitial or pericentromeric positions. As I already mentioned 5S has different evolution and the position differ very frequently.

39 M-FISH multiplex FISH or multicolour FISH

The multicolor FISH uses the combination of several fluorochromes for painting probes of the whole chromosomes. This approach enables us to produce different colors for each chromosome.

40 M-FISH

Multicolour banding (mBAND)

Multicolor FISH has a very high resolution in comparison to G-banding and can detect even very small rearrangements between the chromosomes. This technique also helped to identify the specific position of chromosomes in interphase nuclei. A similar approach, it means the application of several fluorochromes for one FISH but on a single chromosome is called Multicolor banding.

41 Chromosome sorting Microdisection (flow cytometer)

Taq DNA polymerase DOP primers

dNTPs

FISH labeling

DOP-PCR (degenerated-oligonucleotide-primed PCR)

If you want to prepare painting probes you need to obtain the specific chromosome for labeling. You can use a flow cytometer. But for this technique, you need a high mitotic index. You can use also microdissection of selected chromosomes from the surface of the chromosome slides. Only 20 copies enable to obtain the painting probe using DOP-PCR.

42 Pokorná et al. 2015, Chrom. Res.

In this study, the authors used painting probes obtained from Gekko japonicus to test the homology among several other species.

43 danio ZOO-FISH - cross-species chromosome painting, which uses painting human probes specific for whole chromosomes, enables detecting homologous synteny blocks, the occurrence of which is evidence that species share a common ancestry and are related.

mouse chimpanzee

Ancestral karyotype of the genus Sorex Used painting probes of human Ferguson-Smith & Trifonov 2007

Biltueva et al. 2011

The application of painting probes to another species is called Zoo-FISH. You can also buy commercially produced painting probes for chromosomes of human or chicken and test the homology even between more distant organisms. Then you can identify specific chromosomal changes during the karyotype evolution.

44 CGH – comparative genomic hybridization

Sex chromosomes CGH in pachytene of Galleria mellonella female genomic probes were labelled with Alexa Fluor 488 (green) male-derived genomic probes with Cy3 (red) Arrow indicates a region of the W chromosome exclusively stained by female genomic probe. Vítková et al. 2007

Another type of FISH represents CGH – comparative genomic hybridization. This technique was firstly developed for the identification of chromosomal changes in tumor cells in human diagnostics. During this technique, you label the whole genome content of your target (it means all chromosomes) with one fluorochrome. Together with this probe, you make FISH with the genome control probe labeled with another fluorochrome. Then you can see differences between both genomes in color differences. This approach may be either used in tumor cells for identification of the differences between health and damaged cells or for differentiation between males and females.

45 GISH – genomic in situ hybridization - a type of FISH, uses total genomic DNA from one species as the labeled probe and unlabeled genomic DNA from another species at a much higher concentration as blocking DNA, substantially increasing the hybridization specifity

GISH on chromosomes of the water frog Pelophylax esculentus obtained from bone marrow of a single female. B. Metaphase chromosomes hybridized with the Alexa Fluor 488-labeled genomic probe from P. lessonae (green signals); chromosomes were counterstained with PI (red) D. Metaphase chromosomes hybridized with the Cy3-labeled genomic probe from P. ridibundus (red signals); chromosomes were counterstained with DAPI (blue). (Zalesna et al. 2011)

Genomic in situ hybridization (GISH) is a type of FISH that uses total genomic DNA from one species as the labeled probe and unlabeled genomic DNA from another species in much higher concentration as blocking DNA. Then you can visualize the original genomic content in hybrids between the species as it was documented for example in frog Pelophylax esculentus that is in fact hybrid of Pelophylax lessonae and Pelophylax ridibundus.

46 Thank you for your attention

I believe that this brief introduction to the cytogenetic techniques will make you understand how useful they can be in zoology. Thank you for your attention.